This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Nasopharyngeal aspirate (NPA), nasal swab (NS), and throat swab (TS) are common specimens
used for diagnosis of respiratory virus infections based on the detection of viral
genomes, viral antigens and viral isolation. However, there is no documented data
regarding the type of specimen that yields the best result of viral detection. In
this study, quantitative real time RT-PCR specific for M gene was used to determine influenza A viral loads present in NS, NPA and TS samples
collected from patients infected with the 2009 pandemic H1N1, seasonal H1N1 and H3N2
viruses. Various copy numbers of RNA transcripts derived from recombinant plasmids
containing complete M gene insert of each virus strain were assayed by RT-PCR. A standard curve for viral
RNA quantification was constructed by plotting each Ct value against the log quantity
of each standard RNA copy number.

Results

Copy numbers of M gene were obtained through the extrapolation of Ct values of the test samples against
the corresponding standard curve. Among a total of 29 patients with severe influenza
enrolled in this study (12 cases of the 2009 pandemic influenza, 5 cases of seasonal
H1N1 and 12 cases of seasonal H3N2 virus), NPA was found to contain significantly
highest amount of viral loads and followed in order by NS and TS specimen. Viral loads
among patients infected with those viruses were comparable regarding type of specimen
analyzed.

Conclusion

Based on M gene copy numbers, we conclude that NPA is the best specimen for detection of influenza
A viruses, and followed in order by NS and TS.

Background

Influenza A viruses are classified into 16 hemagglutinin (H) and 9 neuraminidase (N)
subtypes [1]. Since the emergence of Russian influenza A (H1N1) in 1977 [2] to the emergence of pandemic influenza A (H1N1) in April 2009, only A/H1N1, A/H3N2
and influenza B viruses have been recognized as human or seasonal influenza. Influenza
virus spreads via respiratory secretion. After an incubation period of about 1-3 days,
the viruses are shed from various kinds of respiratory samples. Upper respiratory
tract specimens, such as nasopharyngeal wash (NPW) or nasopharyngeal aspirate (NPA),
nasal swab (NS), throat swab (TS), endothracheal swab, bronchoalveolar lavage and
tissues, are recommended for virus detection in patients with respiratory tract infection.
These specimens could be used for viral antigen detection, virus isolation and molecular
methods for genome detection. Nevertheless, there is no documented data which addresses
the type of specimen that gives the best yield for the disease diagnosis [3].

Genomes of influenza A and B viruses are composed of 8 negative sense, single-stranded
RNA segments encoded for 10-11 proteins essential for infection and replication [1]. The genomic RNA has been used as targets for amplification by conventional and real
time reverse transcription-polymerase chain reaction (RT-PCR). The highly conserved
M gene-derived primers are usually utilized for diagnosis of all influenza A subtypes,
whereas specific subtype identification targets H or H and N genes. In this study, the protocol established by the U.S., Center for Disease Control
(CDC) for detection of M gene [4] in adjunct with the standard curves of known copies of M RNA transcripts derived either from H1N1, H3N2 or the 2009 pandemic A (H1N1) viruses
was used to quantify the viral loads in specimens collected from patients with severe
influenza prior to receiving anti-viral drug. Our study provided the information on
the clinical specimens that yielded the best diagnostic result; and the viral loads
in patients infected with different influenza subtypes and strains were also compared.

Methods

Subjects and Specimen Collection

This study was approved by the Institutional Review Boards of the Committee on Ethics,
Faculty of Medicine Siriraj Hospital, Mahidol University and the Ministry of Public
Health, Thailand. NPA, NS and TS samples were collected in viral transport medium
(MicroTest™ Multi-Microbe Media; Remel, Lenexa, KS) from patients with severe influenza.
The collection of NPA was performed by flushing through a nasopharyngeal tube with
2 ml of sterile normal saline using a sterile NG-tube or sterile butterfly needle
tube, inserted through the floor of nose. The NPA yield at approximately 0.5 ml volume
was then added with VTM and the 3.5 ml final volume was obtained. The nose and throat
swabbing were performed right after the NPA collection from nostrils and throat, respectively,
using MicroTest™ kit with 3 ml of VTM.

Real time RT-PCR protocols established by CDC as well as viral antigen detection by
QuickVue (Quidel Corporation, San Diego, CA), virus isolation in MDCK cell culture
and serodiagnosis, were used to diagnose influenza virus infection in these patients.
Positive results from at least two diagnostic tests were obtained for each case. A
total of 29 patients enrolled in this study comprised 12 cases of pandemic influenza
A/2009 (H1N1), 5 cases of A/Brisbane/59/2007(H1N1) like- and 12 cases of A/Brisbane/10/2007
(H3N2) like-virus infection. All respiratory specimens were kept at -70°C until tested.

Table 1. Sequences of primers and probes for PCR and real time RT-PCR.

To minimize the test variation, standard curves of M RNA transcripts were constructed in parallel with the detection of viral M RNA in clinical samples in the quantitative real time RT-PCR. The M RNA transcripts were measured by Quant-iT™ RNA Assay Kit (Invitrogen) and diluted
to various copy numbers in a ten folded serial dilution manner; and each known M RNA copy number was assayed by real time RT-PCR according to that described by the
2009 CDC protocol [4]. The sequences of primer and probe sets used in this study are shown in Table 1. A 25 μl reaction mixture of real time RT-PCR comprised 5 μl of total RNA, 12.5 μl
of 2× reaction mix, 0.5 μl of SuperScriptTM III Platinum®Taq Mix (Invitrogen), each 0.8 μM of forward and reverse primers and 0.2 μM of labeled
probe, and H2O was added to bring up the final volume. The amplification was carried out in DNAEngine® Peltier Thermal Cycler with Chromo4™ Real-Time PCR Detector (Bio-Rad Laboratories,
Inc., Hercules, CA) using the amplification cycles of 50°C for 30 min for reverse
transcription, 95°C for 2 min for Taq polymerase activation, followed by 45 cycles of PCR amplification (95°C for 15 sec
and 55°C for 30 sec). Fluorescence signal was obtained at 55°C. The results were analyzed
by MJ OpticonMonitor™ Analysis Software version 3.1 (Bio-Rad). A standard curve was
constructed by plotting each cycle threshold (Ct) value against the log quantity of
standard RNA copy numbers. Total RNA was extracted from the NPA, NS and TS specimens
by QIAamp® Viral RNA Mini Kit (QIAGEN Inc., Valencia, CA) following the manufacturer's instruction.
Real time RT-PCR for detection of influenza A M gene and the RnaseP (RNP) house keeping gene, was carried out. To obtain amount of viral load present
in each clinical sample, the test Ct value was extrapolated against the standard curve
derived from each virus subtype or strain (Fig. 1). The sensitivity of the assay for all 3 subtypes and strain was 100 copies of target
M RNA/real time RT-PCR reaction when the cut-off for positive result was set at 40 cycles.

Figure 1.M transcript standard curve for quantitative detections of the pandemic A/H1N1 (A),
seasonal A/H1N1 (B) and seasonal A/H3N2 viruses (C). The standard curve of M RNA copy numbers was generated by plotting the Ct value (X-axis) against log10 copy numbers of M transcripts (Y-axis). The amount of M copy number in clinical specimens was obtained by extrapolation of the Ct of the test
sample against the standard curve.

Data Analysis

Statistical analysis was performed with SPSS program. Pair t-test was used to compare the mean log10 viral loads among different types of specimens collected from the same subjects and
at the same time. Student t-test was used to analyze the mean log10 viral copy numbers in contemporary specimens from patients infected with different
virus subtypes and strain.

Results and Discussion

Real time RT-PCR protocol was analyzed for its applicability to amplify M genes derived from H1N1, H3N2 and the 2009 pandemic viruses by aligning the primers
and probe nucleotide sequences against those M genes of various influenza subtypes and strains using BioEdit Sequence Alignment Editor
(Fig. 2, Table 2). The forward and reverse primers bound to those M genes with higher than 90% identity, while the probe bound with 100% identity. This
suggested that the CDC primers/probe set can be universally used for detection of
M segments or viral loads of the novel influenza A/2009 (H1N1), seasonal H1N1 and seasonal
H3N2 viruses.

Figure 2.Alignment of M gene fragment from the pandemic A/H1N1, seasonal A/H1N1 and seasonal A/H3N2 viruses
against CDC real time RT-PCR primers and probe sequences. BioEdit Sequence Alignment Editor was used to locate the region of real time RT-PCR
primers and probe binding site within M gene of various subtypes of influenza A viruses.

Table 2. Percentages of identity of primers and probe with the M sequences derived from different virus subtypes and strain.

Three standard curves of M RNA transcripts were constructed with the R2 of 0.996, 0.993 and 0.996 for pandemic A/2009 (H1N1), seasonal H1N1 and seasonal H3N2,
respectively (Fig 1). The M copy numbers per ml of VTM from patients infected with pandemic H1N1 or H3N2 viruses
were significantly highest in NPA samples (pair t-test; P ≤ 0.05) (Table 3). However, number of patients infected with seasonal H1N1 virus was too small for
data analysis. Additionally, viral load levels in patients infected with either subtype
or strain was comparable (student t-test, P > 0.05). M RNAs were detected in all NPA and NS, but not in all TS samples collected from patients
infected with any one of the virus subtypes/strain. The detection rate was shown in
Table 4.

Table 3. Influenza viral loads in various types of clinical specimens collected from patients
infected with different virus subtypes.

RT-PCR for diagnosis of influenza viruses is generally more sensitive than viral isolation
method. The technique detected the viral genome present in dead and alive viruses
including excess viral RNA present in the infected cells; however, virus isolation
detected only live virus particles. RT-PCR is a high through-put and less time consuming
method. In addition, only RT-PCR can differentiate type, subtype and strain of influenza
viruses. Sensitivity of RT-PCR to diagnose the disease not only depends on the protocol,
but also the type of clinical sample used in the diagnosis. Our study has two advantages
that are not commonly conducted in previous reports. Firstly, we had an opportunity
to investigate 3 types of clinical specimens collected from the same individuals at
the same time, e.g., NPA, NS and TS. Secondly, we had employed full length M RNA transcripts derived from A/H1N1, A/H3N2 and the 2009 pandemic viruses to construct
3 standard curves for quantifying viral RNA copy numbers of the contemporary subtype
and strain present in the test specimens, with the assumption that the full length
in vitro M RNA transcripts closely mimics the native structure of the viral M genomic segments. Regardless of viral subtypes and strains (H1N1, H3N2 and 2009 pandemic
H1N1 virus), we found that all NPA and NS specimens were positive for viral genome
detection, while the positive rate was lower in TS specimens.

Previous investigators reported that viral RNA concentration in respiratory samples
and long duration of virus shedding were correlated with influenza disease severity
[6]. Amount and duration of viral shedding are important in the disease treatment and
control of virus spread. Different type of specimens contained different amount of
viral RNA concentration; therefore, using different type of clinical specimens may
yield different information. In addition, there is no reference method for viral load
assay. Peiris et al. [7] reported that viral load in NPA samples of H5N1 patients was lower than those of
H3N2 patients. The finding was further extended by Ward et al. [8] that viral load in throat swab samples of H5N1 patients in 1997 and 2004 was 10-fold
lower than that observed in H3N2 patients, i.e., 1.5 × 106 TCID50/ml versus 1.6 × 105 TCID50/ml (t-test, P < 0.05). On the other hand, de Jong et al. [9] found that viral load in TS from H5N1 patients was significantly higher than that
from H3/H1 patients; and, additionally, TS contained significantly higher H5N1 viral
load than nasal swab samples; meanwhile, viral load in TS and nasal swab samples from
H1/H3 patients was not statistically different. The difference in results obtained
from different groups of investigators might reflect process of specimen collection
and also the different protocols for viral load measurement.

It has been reported that the 2009 pandemic virus preferentially binds sialic acid
receptor with α 2, 6 linkage to galactose (SA α 2,6 Gal), the same as human influenza
H1N1 and H3N2 viruses [10]. Fatality rate in patients infected with the novel virus is less than 1%, except
in that which occurs in patients with underlying conditions, e.g., cardiovascular
disease, hypertension, asthma and diabetes, etc. [11,12]. However, the study in a mammalian model demonstrated that the 2009 pandemic H1N1
virus was more pathogenic than the seasonal H1N1 virus [13]. Our study, therefore, explored the viral load in respiratory secretions collected
prior to anti-viral treatment, and found that the level of viral RNA in cases infected
with the 2009 pandemic H1N1 virus was not statistically different from those infected
with seasonal H1N1 and H3N2 viruses. Mean log10 copies/ml of viral RNA of 7.5-8.0 in NPA, 6.5-7.2 in NS and 4.1-7.4 in TS samples
were found in our study. It is to be kept in mind that all of our patients had severe
influenza at time of specimen collection, and most of them were pediatric patients
(24 children and 5 adults). Duration of viral shedding of the seasonal influenza as
reported by the other groups of investigators was 4-5 days in average [6,14]. A recent report by To et al. [15], showed that the level of the 2009 pandemic viral load of 8 log10 copies/ml was found in respiratory specimens collected before oseltamivir treatment;
and the viral shedding peaked at the day of onset of symptom with median duration
of 4 days [15]. On the other hand, when using plasmid containing amplification target to construct
the standard curve together with using pool of throat and nasal swab as the test samples,
the other study demonstrated that the H1/H3 viral loads of 5.06 ± 1.85 log10 copies/ml were found in patients with major co-morbidities and 3.62 ± 2.13 log10 copies/ml in patients without co-morbidities [6].

Conclusions

Our study suggested that when complete facilities are accessible, such as in clinics
and hospitals, NPA will be the best specimen of choice; and in field investigation,
NS will be the second choice, followed by TS specimen. Using the appropriate specimen
will provide the highest diagnostic rate and the precise strategy for disease treatment
and prevention control.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

PP designed the research study; NN, PN, PK and PhP performed research; NN, PN and
PK analyzed data; NN and PP wrote the manuscript. KK, TC, CS, CC and JF provided specimens.
All authors read and approved the final manuscript.

Acknowledgements

This study is supported by the Thailand Research Fund for Senior Research Scholar
and the South East Asia Infectious Disease Clinical Research Network (SEAICRN), supported
by the US National Institute of Health. NN is supported by Postdoctoral Fellowship
Scholarship, Mahidol University, Thailand. We thank Mrs. Caroline Fukuda and Dr. Steve
Wignall, SEAICRN for their kind coordination.